Verticillium lecanii is an entomopathogen with high potential in biological control of pests. We developed a solid-state fermentation with sugarcane bagasse as carrier absorbing liquid medium to propagate V. lecanii spores. Using statistical experimental design, we optimized the medium composition for spore production. We first used one-factor-at-a-time design to identify corn flour and yeast extract as the best carbon and nitrogen sources for the spore production of V. lecanii. Then, we used two-level fractional factorial design to confirm corn flour, yeast extract, and KH2PO4 as important factors significantly affecting V. lecanii spore production. Finally, we optimized these selected variables using a central composite design and response surface method. The optimal medium composition was (grams per liter): corn flour 35.79, yeast 8.69, KH2PO4 1.63, K2HPO4 0.325, and MgSO4 0.325. Under optimal conditions, spore production reached 1.1∈×∈1010 spores/g dried carrier, much higher than that on wheat bran (1.7∈×∈109 spores/g initial dry matter). © 2009 Springer-Verlag.